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A J Caride

Publications and source records attributed to A J Caride.

At least 19 recordsLinked to original sources
PubMed2001

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Animals↗
PubMed2001

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells.

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.

Animals↗
PubMed1999

The rate of activation by calmodulin of isoform 4 of the plasma membrane Ca(2+) pump is slow and is changed by alternative splicing.

A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca(2+) pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 microM free Ca(2+)) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of full-length 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca(2+) spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca(2+) signals.

Alternative Splicing↗
PubMed1999

Selective decrease of mRNAs encoding plasma membrane calcium pump isoforms 2 and 3 in rat kidney.

BACKGROUND: Although the existence of multiple isoforms of plasma membrane calcium ATPase (PMCA) is now well documented, their biological functions are not yet known. In this study, we set out to investigate the potential role of PMCA isoforms, previously identified in renal cortical tissue, in tubular reabsorption of calcium (Ca2+). METHODS: With use of reverse transcription-polymerase chain reaction analysis, we determined levels of mRNAs encoding isoforms of PMCA1 through PMCA4 in renal cortex, liver, and brain of rats with hypercalciuria induced by feeding with a low-phosphate diet (LPD) as compared with Ca2+-retaining rats that were fed a high-phosphate diet (HPD). RESULTS: We observed that in hypercalciuric LPD-fed rats, the mRNAs encoding isoforms PMCA2b and PMCA3(a + c) are significantly lower (Delta approximately-50%) than in HPD-fed hypocalciuric rats, whereas no changes in mRNAs encoding isoforms PMCA1b and PMCA4 were observed, and mRNA encoding calbindin 28 kDa was increased. On the other hand, the content of mRNAs encoding PMCA2b and PMCA3(a + c) in liver and brain, respectively, was not changed. CONCLUSION: These findings are evidence that expression of PMCA isoforms in the kidney can be selectively modulated in response to pathophysiologic stimuli. The association of a decrease in mRNA encoding PMCA2b and PMCA3(a + c) with hypercalciuria suggests that the two PMCA isoforms may be operant in tubular reabsorption of Ca2+ and its regulation.

Animals↗
PubMed1998

Chemical modification reveals involvement of different sites for nucleotide analogues in the phosphatase activity of the red cell calcium pump.

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Kieosin-Y = 2 microM). FITC treatment increased Kieosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E1, the E1 conformation or the E2 if E1 transition would be involved during calmodulin-dependent pNPPase activity.

4-Nitrophenylphosphatase↗
PubMed1998

mRNA encoding four isoforms of the plasma membrane calcium pump and their variants in rat kidney and nephron segments.

To survey the presence of the four different isoforms of the plasma membrane calcium pump (PMCA) and their alternative splicing variants in the rat kidney, three major zones (cortex, outer medulla, and inner medulla) were macrodissected and probed for the presence of mRNA encoding these isoforms and their variants at the splicing site C by using reverse transcription-polymerase chain reaction (RT-PCR). Both the cortex and the outer medulla showed PMCA 1b, 2b, 3(a and c), and 4b. Semiquantitative comparisons indicated that isoform 2b is more abundant in the cortex than in the outer medulla and conversely, that isoform 3 (a and c) is more abundant in the outer medulla than in the cortex. The inner medulla showed only mRNA for isoforms 1b and 4b. The nephron segments present in the cortex and outer medulla were microdissected and analyzed by RT-PCR. Isoforms 1b, 2b, and 4b were found in all nephron segments but were found more frequently in tubular segments with high rates of Ca2+ reabsorption, suggesting that these isoforms may be involved in transepithelial transport. On the other hand, mRNA encoding isoform 3 (a and c) was most abundant in descending thin limb of Henle but was detected also in glomeruli and cortical thin ascending limb. Its distinct localization suggests that this isoform might have another function, such as in intracellular signalling.

Alternative Splicing↗
PubMed1998

mRNAs coding for the calcium-sensing receptor along the rat nephron: effect of a low-phosphate diet.

We investigated the localization of mRNA encoding the calcium-sensing receptor (CaSR) along the rat nephron. For this purpose, we combined microdissection of nephron segments and RT-PCR techniques. The results indicate that mRNA encoding rat CaSR is present in rat glomeruli and distal segments (medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule and cortical collecting duct), whereas it was not detected in proximal convoluted tubules or proximal straight tubules. We also studied whether the CaSR transcription in kidney cortex was modified in response to low dietary phosphate. No significant changes were detected. Given the fact that a low-phosphate diet increased Ca2+ excretion by more than 50-fold, the results suggest that if the CaSR regulates Ca2+ reabsorption, it does so through receptor occupancy by Ca2+ rather than by changes in receptor expression.

Animals↗
PubMed1997

The membrane topology of the amino-terminal domain of the red cell calcium pump.

A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.

Calcium-Transporting ATPases↗
PubMed1996

Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies.

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

Alternative Splicing↗
PubMed1995

Interaction of unsaturated fatty acids with the red blood cell Ca(2+)-ATPase. Studies with a novel photoactivatable probe.

Unsaturated fatty acids, such as oleic acid, increase both the affinity for Ca2+ and the maximum effect of the Ca(2+)-ATPase of red blood cells [Niggli et al. (1981) J. Biol. Chem. 256, 8588-8592]. With the aim of examining the structural and kinetic details of the interaction between unsaturated fatty acids and the enzyme, we designed and synthesized 8-(5'-azido-O-hexanoylsalicylamido)octanoic acid (AS86), a photoactivatable analogue of unsaturated fatty acids. AS86, interacting noncovalently with the enzyme, shares with oleic acid the following properties: (i) it binds reversibly to the plasma membrane Ca(2+)-ATPase; (ii) in the absence of calmodulin, AS86 shows a biphasic behavior; i.e., at low concentrations it increases the affinity for Ca2+ and the maximum velocity of the enzyme, while at higher concentrations it decreases the maximum velocity; (iii) in the presence of calmodulin, AS86 increases slightly the affinity for Ca2+ and decreases the maximum velocity of the Ca2+ pump; and (iv) AS86 inhibits the activity of the enzyme devoid of its calmodulin-binding domain after proteolysis. When the reagent is covalently bound to the native enzyme, and then activated by calmodulin, increasing amounts of AS86 decrease the maximum velocity along a hyperbolic curve without modifying the apparent affinity for Ca2+. These results could be explained by the eventual existence of two different kind of sites recognizing the reagent: one influencing the affinity for Ca2+ and the other inhibitory of the calmodulin effects. When covalently bound, AS86 exerts its inhibitory effects upon the enzyme lacking the calmodulin-binding domain, thus reflecting that this action is promoted by interaction with a site lying outside this region. The purified enzyme is susceptible to be tagged with 125I-AS86. Both the inhibitory effect on the calmodulin-dependent enzymic activity after covalent binding of AS86 and the photoadduct formation between the enzyme and 125I-AS86 are impaired by the presence of oleic acid in a concentration-dependent fashion. Recognition of photoreactive fatty acid analogues by the purified enzyme could be useful to provide further insight on the location of the interacting sites.

Adult↗
PubMed1995

Unique localization of mRNA encoding plasma membrane Ca2+ pump isoform 3 in rat thin descending loop of Henle.

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.

Animals↗
PubMed1994

Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump.

Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate. Moreover, modification of the pump by SA plus ATP decreased the KCa when the activity was measured in both the absence and presence of calmodulin, suggesting that the residue(s) modified in this case is(are) involved directly in the regulation of the affinity for Ca2+.

Acetylation↗
PubMed1994

Identification of transmembrane domains of the red cell calcium pump with a new photoactivatable phospholipidic probe.

The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophobic photolabeling. Purified Ca2+ pump was reconstituted in asolectin vesicles loaded with [3H]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Soc. 115, 3458-3474, 1993). After photolysis and SDS-PAGE analysis, a significant light-dependent labeling of the Ca2+ pump was found. Controlled proteolysis of the photoadduct with trypsin or protease V8 followed by SDS-PAGE and immunoblotting yielded individual labeled fragments. The labeling pattern indicated the existence of three sequential clusters of transmembrane regions, consistent with the current model for the topography of this enzyme.

Calcium-Transporting ATPases↗
PubMed1993

The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

Biological Transport↗
PubMed1992

Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.

Deletion and truncation mutants of the human erythrocyte Ca2+ pump (hPMCA4b) were expressed in COS-1 cells. The reactivity patterns of these mutants with seven monoclonal antibodies were examined. Of the seven, six (JA9, JA3, 1G4, 4A4, 3E10 and 5F10) react from the cytoplasmic side. JA9 and JA3 reacted near the NH2 terminus and the COOH terminus of the molecule, respectively. 5F10 and 3E10 recognized portions of the large hydrophilic region in the middle of the protein. The epitopes of 1G4 and 4A4 were discontinuous and included residues from the long hydrophilic domain and residues between the proposed transmembrane domains M2 and M3. Antibody 1B10, which reacts from the extracellular side, recognized the COOH-terminal half of the molecule. These results show that the NH2 terminus, the COOH terminus, the region between M2 and M3, and the large hydrophilic region are all on the cytoplasmic side. This means that there are an even number of membrane crossings in both the NH2-terminal and the COOH-terminal halves. Between residues 75 and 300 there must be at least two membrane crossings, and there are at least two membrane crossings in the COOH-terminal half of the molecule.

Animals↗
PubMed1991

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.

4-Nitrophenylphosphatase↗